Effects of Specific Mutations on the Structure and Function of the Copper Protein Amicyanin, a Biological Electron Transfer Mediator

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Health & Medicine

Published on February 18, 2014

Author: BrianDow

Source: slideshare.net


The sole tryptophan of amicyanin, an electron transfer mediating blue copper protein, communicates electronically with the copper ion via excited state energy transfer, which results in fluorescence quenching. The W45Y mutation affects the structure of amicyanin, however it does not affect the function. This means that the tryptophan does not reciprocate electronic communication with the copper. This presentation also discusses preliminary results of the effects of the H95G mutation on amicyanin's ability to mediate electron transfer. The H95G mutation appears to be partially rescued via the addition of imidazole as an external electron transfer mediator.

Effects of Specific Mutations on the Structure and Function of the Copper Protein Amicyanin, a Biological Electron Transfer Mediator Brian Dow Davidson Lab Burnett School of Biomedical Sciences University of Central Florida

Cupredoxin Proteins • Type 1 Copper site Redox Potential (mV) – 2 Histidine, 1 Cysteine, 1 Methionine ligands 800 700 600 500 400 300 200 100 0 • Bacteria, fungi, plants • Mediate electron transfer 680 370 265 184 270 294

Overall Amicyanin Structure Copper

Amicyanin Copper Ligands His95 His53 Met98 Cu(II) Copper Cys92

Cu(II) Amicyanin UV-Visible Absorption Spectra *Cu(I) Amicyanin does not have visible absorption

Copper 10.1 Å Trp45

Trp45 Fluorescence Apoamicyanin (copper removed) Properties of Paracoccus denitrificans amicyanin Mazhar Husain, Victor L. Davidson, and Alan J. Smith Biochemistry 1986 25 (9), 2 431-2436 Amicyanin

Does Trp45 reciprocate electronic communication to the Copper in Amicyanin? ??? Trp45

W45Y Amicyanin

Electronic Properties UV-Visible Absorption & Molar Absorptivity 2.0 Resonance Raman W45Y WT WT W45Y 1.5 Intensity Absorbance 5000 1.0 3000 0.5 0.0 4000 300 400 500 Wavelength (nm) 600 700 2000 200 400 600 Raman shift [cm-1] 800 1000

Kinetic Parameters of W45Y Methylamine Dehydrogenase Amicyanin Cytochrome c551i Complex Copper TTQ Heme

Kinetic Parameters of W45Y MADH-Amicyanin MADHAmicyanin-Cytochrome c551i WT W45Y WT W45Y Kcat 61 ± 2 s-1 66 ± 3 s-1 18 ± 2 s-1 13± 1 s-1 Km 2.3 ± 0.3 μM 3.2 ± 0.5 μM 1.3 ± 0.2 μM 1.0± 0.7 μM

pH-dependent Redox Potential

Redox and pH Dependent Conformational Change His95 Met98 Oxidized Amicyanin Reduced Amicyanin +II +I Cys92 His53

pH-dependent Redox Potential +0.5 pKa in W45Y

Additional H-bond Met51 His95 His53 Met98 Cu(I) Cys92 3.5Å Reduced W45Y Amicyanin Reduced Wild Type Amicyanin *W45Y Cu(I) is less ordered

• Despite fluorescence quenching, there is no effect of Trp45 on copper • Possibility of decreased copper occupancy – Investigate stability of the copper site

Optical Melt 1.0 0.5 0.6 0.4 0.4 W45Y 53.9±0.1° C WT 66.4 ±0.2° C 0.2 0.0 30 Absorbance A595 0.8 0.3 0.2 0.1 40 50 60 70 80 0.0 300 Temperature (°C) When copper site is disrupted, absorbance at 595 nm is lost 400 500 600 Wavelength (nm) 700

Cu(I) Chelation Bathocuproine disulfonate (BCS) Absorbance 0.15 0.10 0.05 0.00 300 400 500 Wavelength (nm) Cu(I) amicyanin has no visible absorbance. Cu(I)-BCS complex 600 absorbs light at 470 nm.

Cu(I) Chelation Normalized A430 1.0 0.8 W45Y 1.5 s-1 WT 0.6 s-1 0.6 0.4 0.2 0.0 0 2 4 Time (min) 6

W45Y Copper Binding • Optical Melt shows 10° C decrease in Cu(II) site stability • Chelation of Cu(I) by BCS is 2.5x faster than WT • Is copper bound less tightly, or is structural stability affected? • Circular Dichroism to better quantitate thermal stability of oxidized, reduced, and apo (copper removed) amicyanin

Structural Thermal Stability Wild Type Amicyanin W45Y Amicyanin

Circular Dichroism Oxidized Reduced Apo WT Tm=60 ±1° C Tm=53 ±2° C Tm=55 ±1° C Tm=46 ±2° C Tm=50 ±1° C Tm=34 ±2° C W45Y

Lost Hydrogen bond Copper Tyr90 2.6Å W45Y Wild Type Amicyanin W45Y Amicyanin

W45Y Summary • Trp45 does not reciprocate electronic communication with the copper • W45Y introduces new H-bond; increases the pH-dependent redox potential; no affect on intrinsic redox potential • W45Y mutation decreases stability by disruption of interior H-bond • Stability is affected regardless of copper presence

Mediation of Biological Electron Transfer by External Chemical Ligands His95 His53 Met98 Cys92

H95G: Type 1 Copper Site Maintained WT WT H95G H95G H95G-Imidazole Extinction Coefficient 4.6mM-1cm-1 -1 4.6mM-1cm 5.6mM-1-1cm-1 5.6mM cm-1 4.7mM-1cm-1 Redox Potential 256 ±1 mV 256 ±1 mV 252 ±1 mV 252 ±1 mV 247±1 mV

Methylamine Dehydrogenase Amicyanin Cytochrome c551i Complex Copper TTQ Heme

Predicted Pathway of Electron Transfer from MADH to Amicyanin His53 His95 Cu(II) TTQ Kinetic and Thermodynamic Analysis of a Physiologic Intermolecular ElectronTransfer Reaction between Methylamine Dehydrogenase and Amicyanin Harold B. Brooks and Victor L. Davidson Biochemistry 1994 33 (19), 5696-5701 Pro94 Carbonyl Met98 Cys92

Electron Transfer Simulation from MADH to H95G Amicyanin His53 Cu(II) TTQ MADH-H95G Simulation Pro94 Gly95 Carbonyl Met98 Cys92

Electron Transfer Simulation from MADH to H95G Amicyanin via Imidazole His53 Imidazole Cu(II) TTQ MADH-Imidazole-H95G Simulation Pro94 Carbonyl Met98 Cys92

Electron Transfer Parameters His53 His53 His53 His95 Imidazole Cu(II)Met98 TTQ Pro94 Cys92 Carbonyl Cu(II) TTQ Met98 Pro94 Gly95 Cys92 Carbonyl MADH-H95G Simulation Met98 Cu(II) TTQ Pro94 Gly95 Cys92 Carbonyl MADH-Imidazole-H95G Simulation MADH-WT Amicyanin KET Kd Distance MADH-H95G Amicyanin MADH-Imidazole-H95G 10 s-1 6 s-1 ±0.4 8 s-1 ±1.0 4.5μM ±0.5 9.0μM ±1.6 4.5 μM ±1.6 7.9Å 12.0Å ±0.5 9.7Å ±0.2

H95G ET Summary • Preliminary data suggest that imidazole can substitute for the His95 side chain • ET is partially restored by the addition of imidazole as a ligand • A new way to probe ET mechanisms and applications – Amino acids, ligand wires, etc.

Acknowledgements • UCF College of Medicine Biomedical Sciences – Victor Davidson – Sooim Shin – Heather Williamson – Yu Tang – Esha Sehanobish • UCF Physics – Suren Tatulian – Alfons Schulte – Jason Matos • Argonne National Laboratories • Seoul National University of Science & Technology – Narayanasami Sukumar – Moonsung Choi

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